Development of an Epidermal Growth Factor Derivative
with EGFR Blocking Activity
Clara Panosa1, Francesc Tebar2, Montserrat Ferrer-Batalle?1, Humphrey Fonge3, Masaharu Seno4,
Raymond M Reilly3,5, Anna Massaguer1*, Rafael De Llorens1
1 Biochemistry and Molecular Biology Unit. Department of Biology, University of Girona, Campus Montilivi, Girona, Catalunya, Spain, 2 Department of Cell Biology,
Immunology and Neurosciences, University of Barcelona, Barcelona, Catalunya, Spain, 3 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy,
University of Toronto, Toronto, Ontario, Canada, 4 Department of Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Okayama,
Japan, 5 Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada
Abstract
The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the
therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB
receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form
of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the
superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR
blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded
peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation.
Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell
growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate
that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory
activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.
Citation: Panosa C, Tebar F, Ferrer-Batalle? M, Fonge H, Seno M, et al. (2013) Development of an Epidermal Growth Factor Derivative with EGFR Blocking
Activity. PLoS ONE 8(7): e69325. doi:10.1371/journal.pone.0069325
Editor: Karl X. Chai, University of Central Florida, United States of America
Received February 28, 2013; Accepted June 9, 2013; Published July 30, 2013
Copyright:  2013 Panosa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study has been supported by a predoctoral grant from the University of Girona awarded to C. Panosa (grant AP2007-01953), by grants from
Generalitat de Catalunya (Spain) (SGR00065), Spanish Ministry of Health (RETICCC ?RD06/0020/0041) and the University of Girona (IdIBGi?PUG2007A/10 ) awarded
to R. de Llorens. This study was also supported by grants BFU2009-13526/BMC and BFU2012-38259 from Ministerio de Econom??a y Competitividad of Spain to F.
Tebar. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Co-author Masaharo Seno is a PLOS ONE Editorial Board member. This does not alter the authors? adherence to all the PLOS ONE policies
on sharing data and materials.
* E-mail: anna.massaguer@udg.edu
Introduction
The cells of multi-cellular organisms need a constant commu-
nication with the environment to maintain their homeostasis, to
survive, to differentiate, and to proliferate. One of the most
important communication systems is represented by the Polypep-
tide Growth Factors that include eight families. The Epidermal
Polypeptide Growth Factor (EGF)/ErbB family represents a very
important ? in fact essential ? family of growth factors governing a
multitude of cellular events. Its importance is also reflected by its
crucial role in many pathologies, most notably in cancer where
they are involved in the sustained chronic proliferation of cancer
cells [1]. The prominent role of the ErbB family in the
development and surviving of cancer cells was described in the
1980?s, when Sporn and Todaro established the theory of the
??autocrine secretion?? of growth factors by cancer cells to maintain a
high rate of proliferation [2]. In many types of cancer the over-
expression of ErbB receptors and enhanced ligand production
allow an autocrine stimulation of the cell proliferation. Since then
an enormous amount of literature has been devoted to the role of
the receptors, but to a lesser extent to the ErbB ligands. After
decades of high interest in the ErbB receptors as therapeutic
targets, and low attention focused on the ligands, new insights have
renewed the importance of the ErbB ligands, especially the EGFR
ligands, as responsible for autocrine/paracrine loops and resis-
tance to old and new therapeutic agents [3?6]. We would like to
suggest that ??it is time to turn again our attention to the role
played by the ligands??, not only the receptors, to design new
therapeutic strategies against cancer.
Human EGF (hEGF) is a small single chain polypeptide
composed of 53 amino acids that adopts a well-defined three
dimensional structure containing three disulfide bonds defining
three loops (namely A, B and C). This scaffold is known as a T-
Knot and is found in a large number of functional unrelated
proteins [7]. EGF structure is tightly related to its function. There
are three contact sites described between the ligand and the EGFR
which in turn is divided by four domains (I, II, III and IV) [8]
(Figure 1): the B loop of EGF interacts with site 1 in domain I, the
A loop of EGF interacts with site 2 in domain III, and the C-
terminal region of EGF interacts with site 3 in domain III. This
structure?function relationship in EGF has been accurately
examined in order to generate antagonistic analogues of EGF,
or EGFR blockers. Some authors have developed shorter synthetic
derivatives of EGF and TGF-a as antagonists of these growth
factors. However, these strategies have not been effective because
the EGF and TGF-a fragments were too short to form the tertiary
PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e69325
structure required for binding to the EGFR [9]. In other studies
the EGF derivative required very high concentrations to exhibit
the desired responses, probably because the short peptide
interacted only with one of the three interfaces of interaction,
namely the site 1 of EGFR [10].
Our group has previously described that the Potato Carboxy-
peptidase Inhibitor (PCI) is an antagonist of human EGF. PCI is a
39-residue protein folded into a 27-residue globular core stabilized
by three disulfide bonds that adopts the same conformation as
EGF, the T-Knot scaffold. This similar structure probably
accounts for the antagonistic activity. PCI competed with EGF
for binding to EGFR, inhibited EGFR activation and cell
proliferation and induced the down regulation of the receptor.
In addition, PCI showed anti-proliferative activity in vitro in several
human cancer cell lines and in vivo in nude mice implanted with a
xenograft of pancreatic cancer cell lines [11,12]. Unfortunately,
PCI?s affinity for EGFR is very low, and high concentrations were
required to achieve the desired inhibitory activity. The structural
and clinical interest of PCI opened the possibility of engineering
new PCI-like EGF antagonists with improved ErbB affinity. The
superimposition of the three-dimensional structures of EGF and
PCI based on disulfide bridge topology [7,11,12], revealed that
PCI lacks the C-terminal part of EGF, one of the important sites
described for interaction with domain III of the receptor. Our
hypothesis was that the lack of this interaction could be responsible
for the described anti-proliferative properties of PCI since EGFR
needs a ligand that binds with high affinity to both domains I and
III to activate the intracellular signalling pathway that promotes
cell division [13,14].
In the present study we describe the production of the human
EGF derivative, EGFt, which lacks the eight final C-terminal
amino acids of EGF in order to preserve the EGF high affinity for
the receptor and the anti-tumour properties of PCI. We describe
the differences between the activation of EGFR by wild type
hEGF or by EGFt, focusing on the key steps of activation pathway:
ligand binding, dimerization, trans-phosphorylation and internal-
ization. We also compare the cell growth promoting effect of both
peptides in order to evaluate the potential application of EGFt as
EGFR blocker for cancer therapy as well as a potential delivery
agent of radio-nuclei conjugates or toxins to cancer cells
overexpressing EGFR.
Materials and Methods
Materials
Restriction enzymes and T4 DNA ligase were all purchased
from New England Biolabs (Ipswich, MA, USA). High-Fidelity
DNA polymerase was from Finnzymes (Espoo, Findland).
Commercial recombinant human Epidermal Growth Factor and
tangential flow filter were purchased from Millipore (Massachu-
setts, USA). The anionic exchange column for HPLC was a
HiPrep DEAE FF 16/10 and the gel filtration HPLC column was
a Superdex 75 10/300 GL, both from GE Healthcare Bio-
Sciences AB (Uppsala, Sweden). Reverse-phase HPLC column
was a Vydac 218TP C18 from Grace (Illinois, USA).
Figure 1. Ribbon diagram of the crystal structure of an EGFR homodimer in complex with two EGF ligands (from pdb code 1IVO). A
The subdomains I, II, III and IV of both receptors are colored pink, orange, cyan and purple, respectively. Held between domains I and III of each
receptor are the EGF ligands colored red and their disulfide bridges yellow. The three main interactions sites between EGF and EGFR are outlined (1, 2
and 3). The interaction site 3 between C-terminal part of EGF and domain III of EGFR is magnified. This binding site is composed by two different
kinds of interactions: hydrophobic interactions between Leu47 (EGF) and Leu382, Phe412, and Ile438 (EGFR), and the formation of hydrogen bonds
with Gln384 side chain of EGFR and carbonyl and amide groups of Gln43 and Arg45, respectively, of EGF. All side chains of residues responsible for
these interactions are shown in different colors depending on the type of interaction. B Ribbon diagram and amino acid sequence of EGF. The C-
terminal part of the molecule is highlighted in black and the side chain of Leu47 is shown in orange in the ribbon diagram. The nucleotides that
encode these 8 amino acids were removed from the encoding sequence to construct the EGF truncated form (EGFt).
doi:10.1371/journal.pone.0069325.g001
An EGF Derivative as EGFR Blocker
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Molecular cloning and expression of hEGF and EGFt on
small batch culture conditions.
hEGF and EGFt encoding sequences were synthesised by
GenScript (Piscataway, NJ, USA). The sequences included the
ompA leader sequence and the enzyme restriction site XbaI on the
59 end of the encoding sequences. Next, the sequences were
digested with the XbaI and BamhI restriction enzymes and ligated
to the expression vector pIN-III-ompA-2 [15] using T4 DNA
ligase. The constructed plasmids, ompA_hEGF and ompA_EGFt,
were transformed into E. coli MC1061 (kindly donated by Dr.
Querol, Autonomous University of Barcelona, Spain). The
positive clones were identified by diagnostic restriction enzyme
digestion and then sequenced to confirm the correct hEGF and
EGFt cloning (ABI PRISM 310 Genetic Analyzer, Applied
Biosystems (Foster City, CA, USA)). E.coli MC1061 cells
containing ompA_hEGF or ompA_EGFt plasmids were grown
at 37uC in 400 ml culture of M9 minimal medium supplemented
with casamino acids (M9 CAS) and with 50 mg/ml ampicillin
(Sigma-Aldrich, St. Louis, MO, USA). After induction with
0.2 mM isopropyl b-D-thiogalactopyranoside (IPTG) (Sigma-
Aldrich) cells were incubated for 24 hours. Next, to determine
the presence of the peptides in the extracellular fraction, the
culture were harvested by centrifugation at 15,0006g for 15 min
and immediately subjected to cellular fractionation to obtain the
different fractions of the culture [16].
Production of hEGF and EGFt in a 10 liter fermentor
OmpA_hEGF or ompA_EGFt MC1061 E.coli positive colonies
were added into a 100 ml Luria-Bertani medium (LB) supple-
mented with 50 mg/ml ampicillin. The culture was grown at 37uC
in a rotary shaker (250 rpm) for a minimum of 5 hours. Then,
5 mL of the culture were inoculated into a flask with 300 mL M9
CAS medium supplemented with 50 mg/ mL ampicillin and
grown at 37uC, over night, in a rotary shaker (250 rpm). The
entire 300 ml grown culture was inoculated in 5000 ml of the same
medium in a 10-L fermentor. The temperature was maintained at
37uC, the partial pressure of O2 at 65% and the pH at 7. The
culture was continuously fed from the 2 early hours of the
fermentation to the 6 h hours with 50% of casamino acids and
glycerol. 1?2 hours later M9 salts (33% of the total volume) and
0.4 mM of IPTG were added into the cell culture. After that, the
culture was continuously fed with M9 salts, oligoelements, and the
other 50% of casaminoacids and glycerol, overnight. 24 hours
later the culture medium was collected. The fermentation was
carried out by the Fermentation Service of the University of
Barcelona using a Biostat B 10L laboratory fermenter (Sartorius
BBI Systems, Postfach, Melsungen, Germany).
Protein purification
The culture medium obtained either by flask or by fermentation
was centrifuged at 100006g and 4uC for 30 min, twice. The
supernatant corresponding to the extracellular medium was then
filtered through 0.4 and 0.2 mm membranes, successively, and
concentrated by tangential flow filtration using a 3000-Da
membrane. The concentrated sample was then dialysed against
50 mM Tris-HCl pH 7.5 (solvent A) to subsequently perform
HPLC (GE Healthcare Bio-Sciences AB: A?KTAbasic) using an
anionic-exchange column (HiPrep DEAE FF 16/10)). The flow
rate was 1 ml/min and the elution buffer (solvent B) was: 50 mM
Tris-HCl, 1M NaCl at pH 7.5. The samples were eluted with a
linear gradient from 0?10% solvent B for 40 min, 10?30% for
80 min and 30?50% for 40 min. Samples containing the
recombinant protein were eluted and collected after 60 min of
elution and lyophilised. In the last purification step, the sample was
dissolved with elution buffer (50 mM Tris-HCl, 200 mM NaCl at
pH 8) and then separated by a gel-filtration HPLC chromatog-
raphy column at a flow rate of 1.2 ml/min. The recombinant
proteins were eluted after about 13 min of elution time.
Protein analysis
The presence of hEGF or EGFt in each production step was
determined on a 16% sodium dodecylsulfate?polyacrylamide gel
electrophoresis (SDS-PAGE). The gel was stained with Coomassie
brilliant blue or analyzed by Western blotting using a primary
rabbit polyclonal antibody against human EGF (Z-12) (sc-275)
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a
secondary antibody coupled to horseradish peroxidase (Dako,
Glostrup, Denmark). The total protein quantity present in the
samples was determined by Lowry-based Bio-Rad assay (Bio-Rad
Laboratories, Hercules, CA, USA). The identity and purity grade
of the proteins were assessed by MALDI-TOF MS and N-terminal
amino acid sequencing (Proteomics and Bioinformatics facility
from Autonomous University of Barcelona, ProteoRed network).
The state of folding of the EGF peptides was analyzed by reverse
phased high performance liquid chromatography (RP-HPLC)
using the methodology described by Chang et al. [17,18] that
allows the identification of minute concentrations of non-native
disulfide scrambling isomers.
Cells and cell culture
The human colorectal adenocarcinoma cell line Caco-2 and the
breast adenocarcinoma cell lines MDA-MB-468 and MCF-7 were
obtained from the American Type Culture Collection (Manassas,
VA, USA). The cells were maintained in Dulbecco?s modified
Eagle?s medium supplemented with 10% fetal bovine serum (FBS)
and 1% penicillin-streptomycin (Gibco-BRL, Grand Island, NY,
USA) at 37uC in a humidified atmosphere containing 5% CO2.
Cell growth and morphology were assessed daily using an inverted
microscope. The cells were routinely maintained for up to 8
passages by successive trypsinization and seeding and the cell
viability was assessed by trypan blue staining (only cultures
displaying .90% viability were used for further work). Possible
contamination with Mycoplasma was routinely checked using the
VenorH GeM Mycoplasma Dection Kit (Minerva Biolabs GmnH,
Berlin, Germany).
Immunofluorescence analysis
The quantification of EGFR expression was performed by flow
cytometry. The cells were analyzed by double immuno-fluorescence
using a mouse monoclonal antibody against human EGFR (GR13
Anti-EGFR (Ab-3)), (Calbiochem, San Diego, CA, USA). The cells
were incubated for 30 min at 4uC with the primary antibody or an
irrelevant antibody as negative control. After washing with
phosphate-buffered saline (PBS) (Gibco-BRL), the cells were
incubated for an additional 30 min at 4uC in the presence of the
Alexa-Fluor 488-conjugated goat anti-mouse IgG antibody (Invi-
trogen, Carlsbad, CA, USA). Next, the fluorescence was analyzed
using a FACSCalibur flow cytometer (Becton Dickinson Immuno-
cytometry Systems, San Jose, CA, USA) equipped with Cell-
QuestTM software (Becton Dickinson). Fluorescence intensity was
represented on a four orders of magnitude log scale (1?10,000). In
each experi-ment 10,000 cells were analyzed.
Receptor binding assay
EGFt was derivatized with diethylenetriamine pentaacetic acid
(DTPA) (Sigma-Aldrich) and radiolabeled with 111Indium-acetate
An EGF Derivative as EGFR Blocker
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(Nordion, Kanata, ON, Canada) to a specific activity of 3.7?18.5
MBq/mg as described [19]. The radiochemical purity of 111In-
labeled EGFt was 95%?98% as assessed by silica gel instant thin
layer chromatography in 100 mmol/L sodium citrate (pH 5). The
receptor-binding properties of 111In-labeled EGFt were evaluated
in a direct radioligand binding assay using MDA-MB-468 human
breast cancer cells (1x106 EGFR/cell [19]). Briefly, various
concentrations of 111In-labeled EGFt (0.6 nM-300 nM) in
120 mL of 150 mM NaCl containing 0.2% bovine serum albumin
(BSA) (Roche, Penzberg, Germany) were incubated with 1x106
cells in 1.5 mL microtubes for 3 h at 4uC. Cell bound radioactivity
was separated from free radioactivity by centrifugation at 2,7006g
for 5 min, and then counted in a c-scintillation counter (Perkin
Elmer, Shelton, CT, USA). Non-specific binding was determined
by conducting the assay in the presence of an excess (30 mM) of
unlabeled EGFt. Specific binding was obtained by subtraction of
non-specific binding from total binding. The equilibrium dissoci-
ation constant value (Kd) was estimated by nonlinear regression of
a plot of the specific binding versus the concentration of 111In-
DTPA-EGFt incubated with the cells using GraphPad Prism
software [20]. The Kd value of
111In-DTPA-hEGF was obtained
from previous work [21].
Western blot analysis
Cells were collected and lysed with ice-cold lysis buffer
containing 20 mM sodium phosphate pH 7.4; 150 mM NaCl;
1% Triton X-100; 5 mM EDTA; 5 mM PMSF; 10 mg/ml
aprotinin and leupeptin; 250 mg/ml sodium vanadate (Sigma-
Aldrich). Protein concentrations were determined by Lowry-based
Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal
amounts of protein extracts were electrophoresed on an 8% SDS-
PAGE gel, transferred to a polyvinylidene difluoride membrane
(Millipore, Bedford, MA, USA) for 3 hours at 100V and then
blocked for 1 h h at room temperature in a blocking buffer
containing 2.5% powdered skim milk in PBS-T (10 mM Tris-HCl,
pH 8.0, 150 mM NaCl and 0.05% Tween-20) to prevent non-
specific antibody binding. Blots were incubated overnight at 4uC
with the corresponding primary antibody diluted in blocking
buffer. After washes with PBS-T, blots were incubated for 1 h with
the corresponding secondary antibody coupled to horseradish
peroxidase (Dako), and revealed with a commercial kit (West Pico
Chemiluminescent Substrate, Pierce Biotechnology, Rockford, IL,
USA). Blots were reprobed with an antibody for b-actin (Santa
Cruz Biotechnology, CA, USA) to control the protein loading and
transfer.
Dimerization analysis
Chemical cross-linking experiments were designed to examine
the ability of hEGF and EGFt proteins to induce receptor
dimerization. The protocol for cross-linking was previously
described by Canals F et al. [22]. MDA-MB-468 cells were
seeded in 100-mm dishes and allowed to grow to 50% confluence.
Cells were harvested in 0.4 ml ice-cold lysis buffer (20 mM sodium
phosphate pH 7.4; 150 mM NaCl; 1% Triton X-100; 5 mM
EDTA and protease and phosphatase inhibitor cocktails (Sigma-
Aldrich)). The protein concentration in cell lysates was determined
by a Lowry-based Bio-Rad assay (Bio-Rad Laboratories, Hercules,
CA, USA). Aliquots of 10 mg of protein were stimulated with
150 nM hEGF or 150 nM and 15 mM EGFt, and additionally
150 nM hEGF together with 150 nM EGFt, at room temperature.
After 30 min incubation, EGFR cross-linking was induced by
addition of 40 mM glutaraldehyde. 1 min later the reaction was
stopped with 0.2 M glycine (pH 9). Next, loading buffer with 5%
b-mercaptoethanol was added to the samples and heated for
5 min at 100uC. The samples were electrophoresed on 5%
polyacrylamide gels (SDS-PAGE), transferred onto PVDF mem-
branes at 30V overnight at 4uC and analysed by Western blotting
as described. Rabbit polyclonal antibodies against human EGFR
(EGFR 1005 (sc-03)) and against HER2 (Neu (C-18)), (both from
Santa Cruz Biotechnology) were used as primary antibodies.
Analysis of EGFR activation and degradation
The ability of EGFt to activate the total phosphotyrosines of the
receptor was determined in MDA-MB-468 cells after treating the
cells with 3 nM, 150 nM hEGF or 150 nM EGFt for 15 minutes
at 37uC. Next, the same amount of cell lysates were analyzed in
parallel by Western blotting with a mouse monoclonal antibody
against total phosphotyrosines conjugated to horseradish peroxi-
dase (PY20) (sc-508 HRP, Santa Cruz Biotechnology) and a
primary antibody against EGFR (EGFR 1005). EGFR specific
phosphoresidues were examined in MDA-MB-468, MCF-7 and
Caco-2 cells after treating the cells with 150 nM hEGFR or EGFt
at 37uC for different incubation times. Samples were analysed by
Western blotting using primary monoclonal antibodies against
phospho-EGFR Tyr 1068 (#2236), Tyr 1173 (#4407), Tyr 1045
(#2237) and Ser 1046/47 (#2238) (Cell Signaling Technology,
Danvers, MA, USA). MAPK and AKT activation was analyzed
with polyclonal antibodies against phosphorylated MAPK
(ERK1/2) and phospho-Akt (Thr308), both from Cell Signaling,
New England Biolabs (Beverly, MA).
For the analysis of EGFR degradation Caco-2 and MCF-7 cells
were incubated at 37uC with starvation medium containing
10 mg/mL of cycloheximide (Sigma Aldrich) and 3 nM, 150 nM
hEGF or 150 nM EGFt for different times. After treatments, cells
were collected and lysed and the different samples were analysed
by Western blotting using the anti-EGFR antibody (EGFR 1005).
EGFR internalization analysis
The capacity of hEGF and EGFt to induce the internalization of
EGFR was determined by a cell-ELISA assay. MCF-7 and Caco-2
cells were seeded in 96-well culture plates (Costar 3596; Corning
Inc., Corning, NY, USA) at a density of 4000 and 5000 cells/well,
respectively. The cells were allowed to attach and grow until they
reached 70% of confluence and then 3 nM hEGF, 150 nM hEGF,
150 nM EGFt, or PBS (control cells) were added. Next, the cells
were incubated for 30 min at 4uC to allow the binding of EGF to its
receptor and then 15 min at 37uC to allow internalization. The cells
were subsequently washed with PBS, fixed with 2% formaldehyde
for 20 min at room temperature and blocked with 1% BSA in PBS
for 1 h at room temperature. After removal of the blocking solution,
the plates were incubated for 1 h at room temperature with the anti-
hEGFR mouse monoclonal antibody (GR13 (Ab-3)) (Calbiochem).
The samples were subsequently washed and incubated for 1 h hwith
a peroxidase-conjugated secondary antibody (Polyclonal Goat Anti-
Mouse Immunogulobulins/HRP; Dako, Glostrup, Denmark).
Briefly, the plates were washed with PBS, and 100 ml of 3,39-5,59-
tetramethyl benzidine (TMB) (Roche, Penzberg, Germany) solution
was added to each well according to the manufacturer?s directions.
The plate was incubated for 5 min at room temperature and then
the enzymatic reaction was stopped by the addition of 100 ml of 1M
H2SO4.The absorbance at 450 nmwasmeasuredwith amicroplate
reader (ELx800; BioTek, Winooski, VT, USA). Each experiment,
including untreated control cells, was carried out in triplicate and the
mean absorbance for each treatment was calculated. The EGFR
present on the cell surface was determined as a percentage of
untreated control cells by dividing the mean absorbance of the three
replicates by the mean absorbance of the untreated control cells.
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Immunofluorescence staining and confocal microscopy
MDA-MB-468 cells grown on coverslips were treated with
150 nM of hEGF or EGFt for different time periods and then
fixed with freshly prepared 4% para-formaldehyde (Electron
Microscopy Sciences, Hatfield, PA, USA) for 12 min at room
temperature and mildly permeabilized with PBS containing 0.1%
Triton X-100 and 0.1% BSA (Roche, Penzberg, Germany) for
3 min at room temperature. Coverslips were then incubated in the
same buffer, in which Triton X-100 was omitted, at room
temperature for 1 h with the anti-hEGFR monoclonal antibody
(GR13 (Ab-3)) (Calbiochem) and a monoclonal antibody against
the nuclear membrane marker Lamin-B1 (A-11) (sc-377000)
(Santa Cruz Biotechnology). Coverslips were washed intensively,
and then incubated with adequate secondary antibodies labelled
with Alexa Fluor 488 and Alexa Fluor 555 (Invitrogen, Carlsbad,
CA, USA), respectively. Both primary and secondary antibody
solutions were precleared by centrifugation at 14,0006g for 10
min. Hoechst (Invitrogen, Carlsbad, CA, USA), was incubated
together with the secondary antibodies to stain the cell nucleus.
Next, the coverslips were mounted in Mowiol mounting medium
(Calbiochem, San Diego, CA, USA). The images were obtained
using a Leica TCS SPS laser-scanning confocal spectral micro-
scope (Leica Microsystems, Wetzlar, Germany). Final analysis of
deconvolved images was performed using ImageJ software (http://
rsb.info.nih.gov/ij/).
Cell proliferation assay
To assess the ability of hEGF and EGFt to promote cell-growth,
Caco-2 and MCF-7 cells were seeded in 96-well plates (5000 and
4000 cells/well, respectively). The optimal cell seeding concentra-
tion was previously established for both cell types, as described in
the supporting information (Fig S1), in order to maintain the cells
in an exponential growth phase along the experiment. The cells
were allowed to attach and grow for 72 h in culture medium, then
the cells were washed and medium without FBS was added to the
cells for 24 h. Next, the cells were treated with concentrations
ranging from 0 to 150 nM of hEGF or EGFt for 96 h for Caco-2
cells and 72 h for MCF-7 cells. The treatments were removed, and
the cells were washed with PBS and incubated for 3 h with 100 ml
of fresh culture medium together with 10 ml of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-
Aldrich, St. Louis, MO, USA). Medium was discarded and
dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA)
was added to each well to dissolve the purple formazan crystals.
Plates were agitated at room temperature for 10 min and the
absorbance of each well was determined with an absorbance
microplate reader (ELx800, BioTek, Winooski, VT, USA) at a
wavelength of 570 nm. Three replicates were used for each
treatment. The cell viability was determined as a percentage of the
untreated control cells, by dividing the mean absorbance of each
treatment by the mean absorbance of the untreated cells.
Statistical analysis
Statistical analysis was performed with the SPSS statistical
software for Windows (version 15.0; SPSS Inc., Chicago, IL,
USA). Quantitative variables were expressed as mean and
standard error (SE). The normality of the data was tested using
the Kolmogorov-Smirnov test. The differences between data with
normal distribution and homogeneous variances were analyzed
using the parametric Student?s t-test, otherwise the non-paramet-
ric Mann-Whitney U test was applied. A value of P,0.05 was
considered significant.
Results
Design of an EGF analogue
Figure 1A shows the ribbon diagram of the crystal structure of an
EGFR homodimer in complex with two EGF ligands. The three
main interaction sites betweenEGF andEGFRaswell as the specific
residues in site 3 involved in the interaction between the receptor
and EGF are indicated. Based on this structure we designed a
truncated EGF analogue, EGFt, lacking the 8 C-terminal amino
acids, including the Leu47 residue important for the hydrophobic
interactions in site 3, in order to generate a peptide with blocking
activity and higher affinity than PCI for the receptor. In figure 1B the
ribbon diagram and the amino acid sequence of EGF are
represented, with the removed C-terminal aminoacids depicted in
black and the Leu47 residue depicted in orange.
Production, purification and analysis of recombinant
hEGF and EGFt
Wild type hEGF was produced in parallel to EGFt in order to
obtain a positive control for the experiments and for future
applications. The encoding sequences of hEGF and EGFt were
cloned into a periplasmic secretion system that contains the OmpA
(Outer membrane protein A) signal sequence [23] in order to
obtain the peptides in the supernatant of the cell culture. After
24 h of culture we confirmed by cell fractionation that hEGF and
EGFt were mainly in the extracellular fraction of the cell culture
(data not shown). The yield of production of these proteins on a
shake flask level was about 1 mg/L of culture. To improve this
yield, the production of the recombinant proteins was next
conducted in a Biostat B 10 L laboratory fermenter under fed-
batch fermentation conditions and the production increased by
25-fold.
Next, the proteins were concentrated from the supernatant and
purified by two chromatographic steps by HPLC: anion exchange
and size exclusion chromatography. Fig. 2A shows the SDS-PAGE
analysis of the samples obtained in the different purification steps
confirming the protein purity in the last step for both peptides.
The identities of both proteins were assessed by MALDI-TOF
analysis confirming their high purity and expected molecular mass:
6222 Da for hEGF and 5094.7 Da for EGFt (Fig. 2B). Additional,
N-terminal amino acid sequence analysis confirmed the correct
amino acid sequence and the proper cleavage of the ompA signal
peptide in the periplasm (data not shown). Fig. 2C shows the
protein folding analysis by reverse-phase HPLC. Both chromato-
grams contain a single peak at the retention time described for
hEGF indicating its correct folding and the absence of folding
scrambles [18].
EGFR expression profile in three EGFR-positive human
tumor cell lines
Before conducting the biological assays, we examined the
EGFR expression levels in MDA-MB468, MCF-7 and Caco-2
cells, with a well-described autocrine loop of TGF-a, by flow
cytometry. As represented in Fig. 3, MDA-MB-468 cells expressed
the highest level of EGFR, while MCF-7 and Caco-2 cells showed
similar moderate expression of EGFR.
EGFR binding affinity of EGFt
MDA-MB-468 cells were used to analyze the binding affinity of
the truncated EGF analogue for EGFR. In Fig. 4, the total,
specific and non-specific binding of 111In-DTPA-EGFt has been
plotted against increasing concentrations of radiolabelled ligand.
The Kd for specific binding of
111In-DTPA-EGFt was calculated
An EGF Derivative as EGFR Blocker
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using a one-site binding hyperbola nonlinear regression analysis by
GraphPad Prism software and indicated that the receptor-binding
affinity of 111In-DTPA-EGFt for EGFR (Kd = 662.03 M61028)
was approximately 46-fold lower than that of 111In-DTPA-hEGF
(Kd = 1.360.26 M61029). For the following experiments, we
decided to use 150 nM nM of hEGF and EGFt, as a saturating
concentration. In some experiments 3 nM of hEGF was also
included since it is the equivalent concentration of the 150 nM
EGFt, based on its binding affinity to the receptor.
EGFR dimerization
MDA-MB-468 cell lysates were treated with either hEGF or
EGFt and in combination for 30 min and then the proteins were
cross-linked by adding 40 mM of glutaraldehyde. The dimer
formation was analyzed by Western blotting. As expected, hEGF
induced EGFR dimers, while no dimerization of EGFR could be
detected after the EGFt treatment (Fig. 5), even when the
concentration was increased to 15 mM (Fig. S2A). These results
suggest that the homodimers were not effectively induced or
stabilized by EGFt stimulation. Moreover, when hEGF and EGFt
were mixed at the same concentration, the EGFR dimer formation
decreased compared to hEGF treatment (Fig. 5), indicating that
EGFt was competing with hEGF for EGFR binding and
competitively decreasing dimer formation. EGFt could neither
induce EGFR-HER2 heterodimerization (Fig. S2B).
EGFR activation
The ability of the recombinant proteins to activate the EGFR
was evaluated by analyzing tyrosine residues phosphorylation after
hEGF and EGFt cell stimulation. MDA-MB-468 cells were treated
Figure 2. Purification and characterization of hEGF and EGFt. A Protein analysis of the different purification steps by Coomassie blue-stained
SDS-PAGE. Molecular weights (M); Lane 1: commercial recombinant hEGF. Lane 2: supernatant of the E.coli fermentor culture before concentration.
Lane 3: 30x concentrated supernatant by tangential flow filtration. Lane 4: product of the first purification step (anionic exchange chromatography).
Lane 5: purified product after the final purification step (gel filtration chromatography). B Determination of the molecular weight of hEGF and EGFt by
mass spectrometry (MALDI-TOF). The analysis confirmed the corresponding molecular weight of the hEGF (6216.6 Da) and EGFt (5087.9 Da). C
Analysis of the state of folding of purified hEGF and EGFt by RP-HPLC. The peaks on the chromatogram correspond to the elution time of well-folded
hEGF.
doi:10.1371/journal.pone.0069325.g002
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with 3 nM, 150 nM hEGF or 150 nM EGFt for 10 min at 37uC.
The cell lysates were analyzed in parallel by Western blotting with
an antibody against total phosphotyrosines and an antibody
against EGFR to identify the bands corresponding to EGFR.
Fig. 6A shows that both recombinant proteins were able to activate
the receptor. However, the phosphorylation induced by EGFt was
markedly lower, even when hEGF concentration was reduced to
3 nM to normalize the binding affinity, indicating an impaired
activation of the receptor.
Next, we compared the effect of EGFt and hEGF on the
phosphorylation of four EGFR specific residues involved in
internalization and proliferation signalling. Fig. 6B shows that
specific residues involved in the internalization of the receptor,
Y1045 and S1046/47, were highly phosphorylated when MDA-
MB-468 cells were stimulated with hEGF. The phosphorylation of
Y1046/47 was similar when the cells were stimulated with EGFt,
while Y1045 was only slightly activated (Fig. 6B, left panel).
Similar results were obtained in MCF-7 and Caco-2 cells (Fig. 6B,
central and right panel). On the other hand, while hEGF induced
a strong phosphorylation of the tyrosines involved in proliferative
signalling pathways, Y1068 and Y1173, EGFt minimally activated
them in any assayed cell line (Fig. 6C). In fact, a decrease in a
proliferation signal output of EGFR, like MAPK (p-ERK1/2) and
PI3K (p-Akt) activation, was observed in EGFt (150 nM nM)
compared to hEGF (3 nM or 150 nM) treatment, being these
differences more evident in MCF-7 and Caco-2 than in MDA-
MB-468 cell lines (Fig. S3). Altogether, the results suggest the
induction of EGFR internalization but a less activation of the
proliferation signals when treating cells with EGFt.
Effect of hEGF and EGFt on EGFR internalization
To further explore the ability of EGFt to trigger the
internalization of EGFR, we developed a cell-ELISA assay using
MCF-7 and Caco-2 cells. In this assay we detected the EGFR that
was not internalized and remained on the cell membrane after
incubation with either hEGF or EGFt for 15 min at 37uC. Fig. 7A
shows the percentage of EGFR on the cell membrane relative to
untreated control cells. In MCF-7 cells, EGFR expression on the
cell membrane was significantly reduced by 21% by EGFt, while
both hEGF treatments induced 50% EGFR internalization.
Similar findings were observed in Caco-2 cells regarding to hEGF,
which induced 34.5% and 45.6% EGFR internalization at 3 nM
and 150 nM, respectively, while the effect of EGFt was more
moderate, leading to 6.3% EGFR internalization.
Figure 3. EGFR expression profile in MDA-MB-468, MCF-7 and
Caco-2 cells. The expression of EGFR on the cell surface was
determined by flow cytometry after indirect immunofluorescence
staining with a monoclonal antibody against human EGFR (solid lines)
followed by incubation with fluorescent-labelled secondary antibody.
An irrelevant primary antibody was used as a negative control (dotted
lines). Histograms were obtained after analyzing 10,000 MCF-7 (red),
Caco-2 (black) and MDA-MB-468 (blue) cells. The fluorescence intensity
is shown on a four-decade log scale.
doi:10.1371/journal.pone.0069325.g003
Figure 4. Representative binding curve for 111In-DTPA-EGFt to
MDA-MB-468 cells. The total binding curve was obtained by
incubating cells with increasing concentration of 111In-labeled EGFt
(0?300 nM). The non-specific binding curve was obtained by adding
increasing concentrations of the 111In-DTPA-EGFt in the presence of
100-fold excess of unlabeled EGFt (30 mM). The specific binding curve
was obtained by subtracting non-specific binding from total binding.
The dissociation constant (Kd) for
111In-labeled EGFt measured in this
assay was 60.0562.03 nM. Each point represents the mean 6 SEM of 3
assays performed in triplicate.
doi:10.1371/journal.pone.0069325.g004
Figure 5. Effect of EGFt on the dimerization of EGFR. Cell lysate
from MDA-MB-468 cells were treated with the indicated concentrations
of hEGF, EGFt or a mixture of both for 30 min. Then the samples were
cross-linked by addition of 40 mM of glutaraldehyde and analyzed by
Western blotting using an anti-EGFR antibody. The position of the EGFR
monomers and dimmers is indicated.
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Effect of hEGF and EGFt on EGFR localization
The cellular localization of EGFR after different times of hEGF
or EGFt stimulation was analyzed by immunofluorescent confocal
microscopy in MDA-MB-468 cells. Triple labelling in Fig. 7B and
7C shows a similar cellular localization of EGFR in both
treatments, hEGF and EGFt respectively. First, in starved
condition and before treatments (time 0), EGFR was almost
completely located in the plasma membrane. After 10 min with
either hEGF or EGFt treatment EGFR were found within the cell
near the cell surface. After 90 min, EGFR was observed
throughout the whole cytoplasm and plasma membrane in both
treatments. Interestingly, after 3 h of treatment a portion of the
receptor was observed in several punctuate staining localized in a
perinuclear region and some punctuate inside the nucleus.
Effect of hEGF and EGFt on EGFR degradation
The ability of EGFt to trigger the lysosomal degradation of
EGFR was also determined. In order to analyze the degradation of
the EGFR, MCF-7 and Caco-2 cells were treated with the protein
synthesis inhibitor cycloheximide and then the total amount of
EGFR was determined by Western blotting after treatment with
hEGF or EGFt for 30, 60, 180 and 300 min. As shown in Fig. 8 a
great proportion of EGFR was degraded after 180 and 300 min
following 3 nM or 150 nM hEGF stimulation. In contrast, close to
100% of the initial receptor level was still intact in all the times
analyzed after EGFt stimulation.
Effect of hEGF and EGFt on cell proliferation
We next compared the ability of EGFt and hEGF, to stimulate
cell proliferation in two human cancer cell lines, MCF-7 and
Caco-2. While hEGF showed a proliferative effect in MCF-7 cells
after 3 days of treatment at a concentrations of 20 and 150 nM,
EGFt did not significantly modify the proliferation rate at any
concentration tested (Fig. 9, left panel). On the other hand, in
Caco-2 cells, after 4 days of treatment, hEGF exhibited a
moderated but significant proliferative effect at any concentration
tested, but EGFt showed a dose-dependent proliferative inhibition
(Fig. 9, right panel). These differences between cell lines were
probably due to the presence of a potent stimulatory autocrine
loop of TGF-a in Caco-2 cells. As Caco-2 cells produce enough
TGF-a to sustain their own proliferation, the addition of hEGF
did not cause a large increase in proliferation. However, when
EGFt was added, it competed with TGF-a for binding EGFR,
causing a decrease in cell proliferation.
Discussion
Signalling through ErbB receptors is of crucial importance in
the control of cell proliferation, survival and differentiation.
Hence, deregulated signalling by these receptors has been
implicated in human malignancies. Recently, EGFR has been
found to be a validated target for cancer chemotherapy to treat
different tumours. However, the marked differences in the clinical
efficiency of different EGFR inhibitors in different carci-nomas, as
well as the development of resistance, have limited the efficacy of
these drugs [3,24,25]. Thus, identifying additional agents that can
Figure 6. Effect of EGFt on the phosphorylation of EGFR. A Analysis of the total phosphorylation of EGFR. MDA-MB-468 cells were treated
with 3 nM, 150 nM hEGF or 150 nM EGFt for 10 min. The whole cell lysates were analyzed in parallel by Western blotting with an antibody against
total-phosphotyrosine residues (PY20) and with an antibody against EGFR (EGFR 1005). B Analysis of the phosphorylation of the C-terminal residues
of EGFR involved in the internalization of the receptor. MDA-MB-468, MCF-7 and Caco-2 cells were treated with 150 nM hEGF or 150 nM nM EGFt for
the indicated periods of time. The whole cell lysates were analyzed by Western blotting with site-specific antibodies for phospho-EGFR tyrosine 1045
and serines 1046/47. C Analysis of the phosphorylation of the C-terminal residues of EGFR involved in the proliferation signaling pathway. MDA-MB-
468, MCF-7 and Caco-2 cells were treated with 150 nM hEGF or 150 nM EGFt for the indicated periods of time. The whole cell lysates were analysed
by Western blotting with site-specific antibodies for phospho-EGFR tyrosines 1068 and 1173. Untreated cells were used as a negative control (Ctl) and
b-actin levels were used as the loading control in Western blotting.
doi:10.1371/journal.pone.0069325.g006
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target EGFR through novel strategies is an important goal in the
treatment of cancer.
The main objective of the present work was to produce a
peptide with EGFR-blocking activity as anti-tumour agent, based
on the PCI, a peptide previously described by our group as an
EGFR blocker, but with a lower affinity for EGFR than EGF. In
order to design a peptide with higher receptor affinity but
preserving the anti-tumour properties of PCI, EGF and PCI
structures were superimposed [7,11,12]. Interestingly, PCI lacks
the C-terminal part of EGF, which is very important for
interaction with the receptor. Thus, we hypothesized that the
absence of this domain could be responsible for the anti-
proliferative activity of PCI. Therefore, we decided to synthesize
an EGF analogue without the C-terminal 8 amino acids,
preserving the last disulphide bridge (Fig. 1). One of the amino
acids removed is Leucine 47, a highly conserved residue,
Figure 7. Effect of EGFt on the internalization and localization of EGFR compared to hEGF. A MCF-7 and Caco-2 cells were treated with
3 nM, 150 nM hEGF or 150 nM EGFt for 30 min at 4uC and then incubated at 37uC for 15 min. The detection of EGFR in the cell membrane was
determined by performing a cell-ELISA assay with specific antibodies. Each column in the graph represents the relative EGFR expression in the cell
membrane versus untreated control cells (CTL) and was the mean 6 SEM of three independent experiments (*P,0.05 vs. control cells). B MDA-MB-
468 cells were exposed for various times to 150 nM hEGF or 150 nM EGFt and stained for EGFR using FITC anti-EGFR antibody (green). The nucleus
and its membrane were stained using Hoescht (blue) and Cy3 anti-lamin B1 antibody (red), respectively. Confocal images were acquired. C The merge
images corresponding to 180 min of treatment were magnified and some slices of a merged xz reconstruction of the stack (slices at 0.3 microns on z
axis) are shown. Arrows indicate green signals within the cell nucleus. Scale bar, 10 mm.
doi:10.1371/journal.pone.0069325.g007
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important for EGF binding to the EGFR and its mitogenic activity
[26?28].
We had previously reported the production of hEGF by
refolding it from E.coli inclusion bodies [16]. However, the
recovery of well folded and bioactive proteins from inclusion
bodies involves complicated and costly de-naturation and refolding
processes [29]. In the present work we have cloned and expressed
both the wild type human EGF and the new truncated EGF
peptide (EGFt) using an extracellular expression system that
simplifies the purification protocol to only two chromatographic
steps and produces the recombinant proteins with their correct
folding, a really important feature because the function of this
peptide is tightly related to its 3D structure. Furthermore, we have
described a novel production methodology by fermentation that
increases several times the yield obtained in small batch shake flask
production. The enhanced protein production allowed the
performance of the in vitro assays that addressed the differences
between the activation of the EGFR pathway by wild type hEGF
and by EGFt in the key EGFR activation steps: receptor binding,
dimerization, trans-phosphorylation and internalization.
The affinity of EGFt for EGFR was determined in order to
assess if it was increased compared to PCI. As expected, EGFt had
a dissociation constant (Kd) which was lower but in the same order
of magnitude as hEGF (nM), while for PCI the effective
concentration was one order of magnitude higher (mM) [11,12].
Thus, the adopted approach significantly increased the affinity of
the peptide for the EGFR. Although EGFt showed specific binding
and high affinity for EGFR, the affinity of the receptor for EGFt
was lower than for the wild type hEGF. This result was also
expected because the binding of EGF to its receptor relies mainly
on three contact sites (Fig. 1A) and EGFt lacks the Leu47 residue
which is of great importance for one of the interactions in site 3 of
the EGF-EGFR binding interfaces (Fig. 1B). Binding affinity
experiments allowed us to establish the working concentrations for
the subsequent experiments.
EGFR dimerization was detected only when cell lysates were
activated with hEGF, but not with EGFt. The binding of EGF
Figure 8. Effect of EGFt treatment on EGFR degradation. A MCF-7 and Caco-2 cycloheximide treated cells were incubated with 3 nM, 150 nM
hEGF or 150 nM EGFt at 37uC for different time periods as indicated. Cells were lysed, and the amount of EGFR was determined by Western blotting.
Actin levels were used as loading control. B Levels of EGFR were determined by densitometry and normalized versus actin levels. Each column
represents the mean of two replicates 6 SEM.
doi:10.1371/journal.pone.0069325.g008
Figure 9. Effect of EGFt on the growth of two human cancer cells. MCF-7 and Caco-2 cells were incubated for 72 h or 96 h h in a culture
medium without FBS supplemented with 10, 20 and 150 nM hEGF or EGFt. The cell proliferation was assessed by MTT assays. Each column in the
graph represents the relative cell proliferation versus untreated cells (control) and was the mean 6 SEM of three independent experiments (*P,0.05
vs. control cells).
doi:10.1371/journal.pone.0069325.g009
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through the three interaction sites of EGFR shifts the equilibrium
between the two distinct EGFR conformations to the untethered
one, enabling the dimerization arm in domain II to interact with
an identical dimerization arm of another receptor to form the
dimer [13,14]. The differences between dimerization induced by
hEGF versus EGFt might be related to the ability to stabilize the
untethered configuration. As previously mentioned, EGFt do not
interact with all three interaction sites required to promote the
dramatic domain rearrangement that will shift the EGFR
ectodomain into an untethered configuration in which its
dimerization arm is exposed. In agreement, some authors have
previously proposed that a ligand that competes effectively with
EGF for EGFR binding, but which fails to promote the domain
rearrangement, would be a potent antagonist, and also predicted
that a monovalent ligand that binds with very high affinity to only
domain I or domain III (but not both) would fulfil these criteria
[13]. Moreover, recent understanding of the mechanism of action
of suramin as a growth factor blocker with anti-cancer activity
supports this idea. Suramin binds to EGFR by its C-terminal
domain, and the authors suggested that suramin prevents hEGF
from interacting with EGFR at site 3 and, as a consequence,
EGFR can not undergo the proper conformational change to
dimerize and initiate signal transduction [30].
The effects of EGFt in the trans-phosphorylation of EGFR were
investigated, assessing the ability of EGFt to initiate downstream
signalling pathways. The results indicated that EGFt is able to
stimulate EGFR phosphorylation. However, the phosphorylation
level induced by EGFt was not as high as hEGF, even at
concentrations greater than its Kd. These results show that even
though it is not possible to detect EGFR dimerization, EGFt is still
causing EGFR trans-phosphorylation. This indicates that EGFt
was likely inducing a small EGFR conformational re-arrangement
that could exposes the dimerization arm in part, but probably not
sufficient to stabilize the dimer. Consequently, the kinase domain
was not fully activated to proper trans-phosphorylate all tyrosine
residues.
It has recently been postulated that EGF family members that
bind the same receptor are able to stimulate different biological
responses both in cell culture and in vivo, due to distinctions in the
conformation of the ligand-bound receptor and subsequent
differences in the sites of receptor tyrosine phosphorylation
[31,32]. Moreover, it has been demonstrated that the structure
of the EGFR extracellular domain dimer is distinct when bound to
EGF or TGF-a. Thus, ligand-specific differences in the interaction
with the receptor may lead to differences in the receptor tyrosine
residue availability for the receptor kinase domain [32]. EGFt may
induce a distinct EGFR ectodomain conformation than hEGF
and, as a consequence, EGFt could be stimulating different
downstream responses. Different tyrosine and serine residues were
analyzed to address the phosphorylation activation status of two
main cellular responses: proliferation and down-regulation of
EGFR by internalization of the receptor. Receptor down-
regulation is the most prominent regulatory system for EGFR
signaling attenuation and involves the internalization and subse-
quent degradation of the activated receptor in lysosomes. This
molecular event mainly involves phosphorylation of Tyr-1045 and
Ser-1046/7 of EGFR [33?5]. As expected, hEGF strongly induced
the phosphorylation of both residues while EGFt did it moder-
ately, indicating that EGFt could be inducing some EGFR
internalization thus promoting some EGFR pathway down-
regulation. Regarding cell proliferation, signalling by EGFR
involves activation of the downstream MAPK/ERK signalling
pathway, which is triggered by the phosphorylation of Tyr-1068
and Tyr-1173 [28]. The analysis of the Tyr-1068 and Tyr-1173
phosphorylated residues after activation with either hEGF or
EGFt revealed that while hEGF strongly stimulated their
phosphorylation, EGFt hardly stimulated them compared to
untreated cells. This result indicated that EGFt might not be
activating cell growth, a similar result that was observed using PCI
[11,12].
When investigating the effects of EGFt at the cellular level, we
found that it was able to induce EGFR internalization, although in
a much lesser extent than hEGF, what is in agreement with the
reduced phosphorylation of residues involved in down regulation
of EGFR. It is known that activated EGFR traffics from the
plasma membrane to the cytoplasm through endocytic vesicles.
Then, internalized EGFR can be eventually degraded in
lysosomes, or recycled to the plasma membrane. Moreover,
several reports have identified additional intracellular destinations
for the internalized receptor, including the nucleus [36?38]. Our
confocal microscopy analysis revealed that the localization of the
receptor after its activation with either hEGF or EGFt was similar
in both cases. Interestingly, after 3 hour of induction, a small
amount of EGFR staining was observed into the cell nucleus.
Different studies have postulated that intranuclear EGFR may act
as a transcriptional co-activator for various oncogenic genes
[36,37]. The study of EGFR degradation revealed that while
hEGF stimulated EGFR degradation, EGFt did not. These results
suggested that when EGFt stimulated EGFR internalization the
receptor was not extensively routed to lysosomes. We hypothesize
that these differences in ligand-receptor processing between hEGF
and EGFt could be similar to what some authors have observed
before with TGF-a. The EGF binding to EGFR is relatively more
stable at the pH of endosomes, so upon EGF binding to EGFR it
can be transported to lysosomes. In contrast, TGF-a rapidly
dissociates from the receptor when exposed to the low pH of
endosomes, and the receptor and ligand dissociate and are
recycled back to the plasma membrane [39,40]. If EGFt behaves
as TGF-a with respect to internalization, it means that EGFt may
be recycled back to the cell surface, where it can exert its blocking
activity on the EGFR again. A similar result was observed with
PCI [11,12].
Regarding the effect of EGFt on cell proliferation, we observed
that EGFt did not stimulate the proliferation of either tested cell
lines. Moreover, a different cell proliferation response was detected
between MCF-7 and Caco-2 cell lines treated with EGFt. While in
MCF-7 EGFt did not stimulate cell growth at any of the
concentrations tested, in Caco-2 cells EGFt inhibited about 20%
of cell growth, compared to untreated control cells. Caco-2 cell
line has a powerful autocrine loop of TGF-a and its growth
depends on it [41?43]. These results indicate that EGFt inhibits
ligand autocrine mitogenic activity by competing with TGF-a. So
that, the data suggest that EGFt is able to inhibit the in vitro growth
of those cancer cell lines that depend on an autocrine induction of
cell proliferation by secretion of EGFR ligands.
In conclusion, we have demonstrated that EGFt binds to EGFR
but does not stabilize the dimer formation, leading to an impaired
EGFR trans-phosphorylation on tyrosine residues involved in
proliferation signalling. Interestingly, EGFt induces the internal-
ization of the receptor and presumably promotes the translocation
of the receptor to the cell nucleus. Furthermore, EGFt competes
with EGFR native ligands, inhibiting the proliferation of cells with
an autocrine growth control via EGFR, acting as an inverse
agonist or EGFR blocker. These findings indicate the importance
of the hydrophobic interaction of EGF with EGFR on site 3
interface to activate the mitogenic EGFR signalling, and open up
the possibility to insert new amino acid changes that could increase
EGFt affinity for EGFR and its blocking activity.
An EGF Derivative as EGFR Blocker
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Moreover, EGFt could also be used as a targeted toxin delivery
agent. In this respect, we are currently developing 111In?DTPA?
EGFt as a potential Auger electron-emitting radiotherapeutic for
EGFR-positive breast cancer. The Auger electrons emitted by
111In in the nucleus of EGFR-positive breast cancer cells by 111In-
DTPA-hEGF were shown to provide potent cytotoxic effects in
vitro and tumour growth-inhibition in vivo [44,45]. EGFt would
have advantages over hEGF for constructing such a radiothera-
peutic agent due to its lack of EGFR-mediated growth-stimulatory
activity.
Supporting Information
Figure S1 Assessment of the optimal cell concentration
for cell proliferation assays. To determine the optimal initial
seeding density, different cell concentrations (1000, 2000, 3000,
4000, 5000, 6000, 7000 and 8000 cells per well) were plated into
96-well plates (6 replicates per cell concentration) and incubated as
described in the proliferation assay. The cells were allowed to
attach and grow for 72 h, starved for 24 h and then treated with
the highest concentration of hEGF used in the proliferation assay
(150 nM) for 72 h (MCF-7 cells) or 96 h (Caco-2 cells). Finally, the
proliferation of the cells was determined by an MTT assay and the
main absorvance was represented versus the number of seeded
cells. 5000 Caco-2 and 4000 MCF-7 cells/well were selected as
optimal initial cell concentrations as they lied within the linear
portion of the plot, indicating that cells were still in an exponential
growth rate at the end of the experiment.
(TIF)
Figure S2 Effect of EGFt on the dimerization of EGFR. A
Cell lysates from MDA-MB-468 cells were treated with the
indicated concentrations of hEGF, EGFt or a mixture of both for
30 min. Untreated cells were used as control. Then the samples
were cross-linked by addition of 40 mM of glutaraldehyde and
analyzed by Western blotting using an anti-EGFR antibody. The
position of the EGFR monomers and dimers is indicated. B To
examine the effect of EGFt on EGFR heterodimerization with
HER2, MDA-MB-468 cells were treated with 150 nM hEGF,
150 nM EGFt or medium alone as control. After performing the
dimerization assay, the samples were analyzed by Western blotting
using antibodies against EGFR (left panel) and HER2 (right
panel). The position of the EGFR monomers and dimers is
indicated.
(TIF)
Figure S3 Comparative effect between hEGF and EGFt
on MAPK and Akt activation in MCF-7, Caco-2 and MDA-
MB-468 cells. Serum starved MCF-7 (A) Caco-2 and MDA-MB-
468 (B) cells growth in 6 well plates were stimulated with 3 nM,
150 nM hEGF or 150 nM EGFt as specified for the period of time
indicated at 37uC. Lysates with equal amount of protein were
electrophoresed and phosphorylated EGFR (p-Tyr), MAPK (p-
ERK1/2) and Akt/PKB (p-Thr308) were analyzed by Western
blotting. Actin detection was used as a loading control. Figure
shows one representative experiment from duplicate samples.
(TIF)
Acknowledgments
The authors would like to thank Dr. Rosa Peracaula for her critical reading
and valuable discussions of the manuscript. We thank Dra. Maria Calvo,
Anna Bosch and Elisenda Coll for assistance in the confocal imaging
(Unitat Microscopia Confocal, Serveis Cientificote`cnics, Universitat de
Barcelona-IDIBAPS) and Maria Molinos for technical assistance.
Author Contributions
Conceived and designed the experiments: CP FT RMR AM RDLL.
Performed the experiments: CP MFB HF. Analyzed the data: CP FT HF
MS RMR AM RDLL. Contributed reagents/materials/analysis tools: FT
RMR MS. Wrote the paper: CP FT AM RDLL.
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